HCMV US28 regulation of host cell signaling in viral latency and reactivation

摘要-项目3 人巨细胞病毒（HCMV）是一种b-疱疹病毒，感染44-100%的人群，并且仍然是一种高致病性病毒。 实体器官移植（SOT）和异基因造血干细胞移植中发病率和死亡率重要原因 细胞移植（SCT）受者。SCT患者的感染通常与骨髓抑制和移植相关。 失败是由于病毒从潜伏期重新激活，但相关的机制仍然在很大程度上是未知的。HCMV 编码多种潜伏期相关基因产物，包括趋化因子受体US 28，其结合CC- 趋化因子以及CX 3C-趋化因子Fractalkine。我们已经证明US 28配体依赖性 需要信号传导来维持病毒在体外CD 34+造血祖细胞（HPC）和体内的潜伏期 在人源化小鼠中。US 28在两种系统中都需要用于再活化潜伏的HCMV并促进CD 34 + HPC分化为髓系细胞，表明US 28在这两个过程中起着动态作用。而 我们已经确定US 28的配体结合活性是HCMV潜伏期所必需的，我们还不知道是否 这种效应是配体特异性的。此外，我们最近已经证明，HCMV编码的趋化因子配体， 调节US 28信号传导。我们假设宿主CC趋化因子和病毒趋化因子 UL 146/UL 147通过激活特定的信号通路促进潜伏期，而其他US 28配体有助于直接 通过修改信号重新激活。我们假设US 28配体结合活性（配体特异性）是必需的， 通过与EGFR信号传导介导维持潜伏期，并且在再活化期间，US 28拮抗EGFR信号传导 并转变为激活RhoA通路以增强生产性复制。在具体目标1中，我们将确定 其中US 28配体使用针对宿主的中和抗体在潜伏期和再活化期间重编程细胞， 以及病毒趋化因子、影响US 28选择性结合配体的能力的病毒突变体和病毒突变体 缺乏病毒趋化因子在SA 2中，我们将利用使用BirA-Turbo生成的US 28相互作用组数据 接近传感器技术，以识别在延迟和重新激活中起作用的信号因素。通过 通过突变分析，我们鉴定了US 28的特定区域，在C '末端尾和胞内环3中 域，其修改US 28信令。我们将确定这些突变对US 28和US 29的影响。 使用体外和体内模型研究相互作用组、潜伏期和再激活。最后，在SA 3中，我们将定义如何 US 28信号传导影响其他HCMV蛋白（项目1和4）和miRNA（项目2）， 关于US 28如何与EGFR通路交叉，以促进EGFR的联合HCMV操纵 信令（项目5），因此控制延迟和重新激活的总体目标， 提议这项研究的结果将产生新的病毒潜伏期和再激活模式，促进 开发新的治疗方法以防止病毒再活化。

SUMMARY - PROJECT 3 Human cytomegalovirus (HCMV) is a b-herpesvirus infecting 44-100% of the population and remains a significant cause of morbidity and mortality in solid organ transplant (SOT) and allogeneic hematopoietic stem cell transplant (SCT) recipients. Infection in SCT patients is often associated with myelosuppression and graft failure due to virus reactivation from latency, but the associated mechanisms are still largely unknown. HCMV encodes multiple latency-associated gene products including the chemokine receptor US28, which binds CC- chemokines as well as the CX3C-chemokine Fractalkine. We have demonstrated that US28 ligand-dependent signaling is required to maintain viral latency in vitro in CD34+ hematopoietic progenitor cells (HPCs) and in vivo in humanized mice. US28 was required in both systems for reactivation of latent HCMV and promotes CD34+ HPC differentiation into myeloid lineage cells, indicating that US28 plays a dynamic role in both processes. While we have identified that ligand binding activity for US28 is required for HCMV latency, we do not yet know whether this effect is ligand-specific. In addition, we have recently demonstrated that HCMV-encoded chemokine ligands modulate US28 signaling. We hypothesize that both the host CC chemokines and viral chemokines UL146/UL147 promote latency by activating specific signaling pathways while other US28 ligands help direct reactivation by modifying signaling. We hypothesize that US28 ligand binding activity (ligand-specific) is required to maintain latency by interfacing with EGFR signaling and during reactivation US28 antagonizes EGFR signaling and shifts to activating RhoA pathways to enhance productive replication. In Specific Aim 1, we will determine which US28 ligands reprogram cells during latency and reactivation using neutralizing antibodies against host and viral chemokines, viral mutants that impact the ability of US28 to selectively bind ligands, and viral mutants lacking the viral chemokines. In SA2, we will utilize our US28 interactome data generated using the BirA-Turbo proximity sensor technology to identify signaling factors that play a role in latency and reactivation. Through mutational analyses we have identified specific regions of US28, in the C’terminal tail and intracellular loop 3 domains, that modify US28 signaling. We will determine what effect these mutations have on both the US28 interactome and latency and reactivation using in vitro and in vivo models. Lastly, in SA3, we will define how US28 signaling influences other HCMV proteins (Projects 1 and 4) and miRNAs (Project 2) with specific focus on how US28 intersects with the EGFR pathway, to contribute to the combined HCMV-manipulation of EGFR signaling (Project 5) and therefore control of latency and reactivation within the overarching goals of this proposal. Results of this study will generate new virus latency and reactivation paradigms promoting the development of novel therapies to prevent virus reactivation.